Extracellular ATP has been shown to affect the function of numerous types of intact cells. In the case of smooth muscle, in particular, considerable evidence exists to support a physiological neurotransmitter role for ATP. Significantly, recent studies by the applicant have demonstrated that micromolar concentrations of extracellular ATP can activate rapid increases in cytosolic (Ca2+) in the DDT-1 smooth muscle cell line derived from a leiomyosarcoma of hamster vas deferens. This mobilization of Ca2+ by extracellular ATP is very similar to that elicited by alpha1-adrenergic receptor agonists in this cell line; additional studies have shown that these latter receptors are coupled to the inositol phospholipid signalling system. The experiments described in this proposal will characterize in detail the transmembrane signalling actions of extracellular ATP in the DDT1 cell line with particular emphasis on inositol phospholipid metabolism and modulation of cytosolic (Ca2+). Comparison of the effects triggered by ATP with those triggered by alpha1-adrenergic receptor agonists will provide strong evidence for or against the existence of a cell surface receptor for ATP which is coupled to the inositol phospholipid signalling cascade. Additional experiments will be directed towards: 1) characterization of the stereo-specificity of the putative ATP receptor; 2) evaluation of the possible relationships between the signalling actions of extracellular ATP and the various ecto-enzymes which utilize ATP; 3) determination of the possible role of extracellular ATP in regulating motility and phosphorylation of regulatory proteins in DDT1 cells; and 4) characterization of the possible signalling role of extracellular ATP in primary cultures of visceral and vascular smooth muscle. The proposed studies will utilize fluorometric and isotopic techniques for monitoring cytosolic (Ca2+) and pH, as well as established protocols for measuring rapid changes in phospholipid turnover, nucleotide metabolism, ion fluxes, and protein phosphorylation. The results derived from these studies will provide strong evidence for judging whether ATP can be added to the rapidly growing list of Ca2+-mobilizing hormones, growth factors, and neurotransmitters. Such a finding would constitute a significant contribution to our current understanding of the physiology and and pathologgy of receptor-mediated signal transduction in both smooth muscle and other tissue types.